Maxiprep

1I maxi-preped in order to use for ligation. (Part 2H discovered to be faulty on Wednesday).

Miniprep

Minipreped J37016 R colonies 1, 2, 3, 4

Gels show that colony 2 and 4 were successful

Cultured up colony 2 for maxiprep tomorrow

Missing Maxis!!

Cultured up parts 12D and 5M in order to maxi first thing tomorrow.

Media

Made up 2 batches of 200 mL M9

40 mL 10x M9 salts

12 mL 10 mg/mL Thiamine

4 mL 40% glycerol

8 mL 10% casamino acids

8 mL 0.1M MgSO4

80 μL 0.5 M CaCl2

Make up to 400 μL with sterile water

Filter sterilise the entire batch (see Sue)

Kept in fridge for use

Made up 2 batches of 500 mL LB

10 g Tryptone

5 g Yeast Extract

10 g NaCl

Make up to 1 L with distilled water

Take to autoclave

To do tomorrow

Miniprep

4G - 2 colonies

1M - 2 colonies

J37015RS - 4 colonies, more colonies to ensure we have the final construct that we want, then take to sequence if we do

2H (2nd attempt) - 2 colonies, to check if the DNA is actually wrong in the parts registry

Maxiprep

J37016, then take to sequence

12D - missing maxiprep???

5M - also missing maxiprep???

Please find the concentrations of the DNA by taking to Dr. Jensen on 5th floor of biochem building

For sequencing - for tomorrow

Use total of 12.5 uL in a 0.5 mL eppendorf tube

1 μg of DNA - you will need the concentrations

12.5 pmol primer

Make up to 12.5 μL with ultrapure water

Take to either Dr. Mann or Dr. Jensen to sequence

2H

Because we have established that there is a problem with the Spe1 restriction site on our 2H culture and as this is now holding up our progress, we have decided to change our ligations steps. It will increase the number of ligations we are doing by one, but the time that this takes will not be increased, as we are able to do two of these ligations in parallel.

Tomorrow we will ligate parts 12D and 9G together and parts 1I and J37019 together

(1I + 9G are the components that 2H is made up of)

We can then ligate the two ligated parts together.

If all goes to plan this test construct will be complete by this time next week